Composition containing d-cycloserine and d-alanine for memory and learning enhancement or treatment of a cognitive or psychotic disorder

ABSTRACT

A composition is described for use in memory and learning enhancement or for treatment of a cognitive disorder or a psychotic disorder. This composition contains the compound D-cycloserine and D-alanine and provides reduced adverse side effects typically associated with chronic D-cycloserine use.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.07/324,279 filed Mar. 15, 1989, now abandoned.

FIELD OF THE INVENTION

This invention is in the field of clinical neurology and relatesspecifically to compounds, formulations and methods for memoryenhancement and for treatment of cognitive and psychotic disorders.

BACKGROUND OF THE INVENTION

There are many memory-related conditions for which therapeutictreatments are under investigation, such as methods to enhance memory orto treat memory dysfunction. For example, memory dysfunction is linkedto the aging process, as well as to neurodegenerative diseases such asAlzheimer's disease. Also, memory impairment can follow head trauma ormulti-infarct dementia. Many compounds and treatments have beeninvestigated which can enhance cognitive processes, that is, which canimprove memory and retention.

For example, the compound D-cycloserine has been discovered recently toprovide improvements in cognitive function and to be useful in treatmentof cognitive dysfunction, as described in U.S. Pat. application Ser. No.07/127,121 filed Dec. 1, 1987, now U.S. Pat. No. 4,904,681 andapplication PCT/US88/04244 filed Dec. 1, 1988.

There are many psychotic states for which therapeutic treatments areunder investigation. Drugs which are currently available on the marketare thought to act as antagonists at the dopaminergic receptors locatedin the Central Nervous System (CNS), examples of such drugs beinghaloperidol and chlorpromazine. These drugs typically induce longlasting and sometimes irreversible side-effects, such as tardivedyskinesia. Thus, the search for improvements in therapy for psychoticdisorders has been directed to use of drugs with a different mode ofaction.

Phencyclidine [1-(-phenylcyclohexyl)piperidine; PCP] is a known generalanesthetic and is in use as an animal tranquilizer. PCP is a potentpsychotomimetic agent used frequently as a "street" drug. Widespreadabuse of PCP has led to increased incidence of PCP-induced psychoses [C.V. Showalter et al, Amer. J Psychiat., 134, 1234 (1977)]. PCP abusersexperience an apparent sensory isolation accompanied by a feeling ofdepersonalization which can be terrifying to the person. Thesesubjective changes make PCP an appropriate drug model for study ofschizophrenia. The most impressive evidence that PCP psychosis resemblesschizophrenia is the fact that drug users have been mistaken byexperienced psychiatrists for schizophrenics before obtaining thehistory of drug use [S. H. Snyder, Nature, 355-356 (1980)].

PCP has been reported to modulate allosterically the NMDA receptor [P.Loo et al, Eur. J. Pharmacol., 467-468 (1986)] and it has beenspeculated that the psychotomimetic activity of PCP is related to itsantagonism of NMDA transmission [C. A. Tamminga et al, Synapse, 1,497-504 (1987)]. Facilitation of NMDA transmission by action at theglycine modulatory site may antagonize the effect of an endogenousPCP-like ligand [R. Quirion et al, Peptides, 5, 967-973 (1984)]. Also ithas been postulated that glutamatergic action at the glycine-modulatedNMDA receptor may be a route to treatment of schizophrenic [S. I. Deutchet al, Clin. Neuropharm., 12, 1, 1-13 (1989)].

D-cycloserine has long been known as a bacteriostatic agent [see TheMerck Index, Monograph No. 2747, 10th Edn., Merck & Co., p.395 (1983)].Its mechanism of action is believed to involve inhibition of cell wallsynthesis in susceptible organisms by competing with D-alanine forincorporation into the bacterial cell wall. Also, it is known that thein vitro antibacterial activity of D-cycloserine may be inhibited withD-alanine [Goodman & Gilman, The Pharmacologic Basis of Therapeutics,7th Edn., MacMillan, N.Y., p. 1209 (1985)].

The compound D-cycloserine, in its D- and L-isomer forms, has also beenevaluated for CNS effects in animals [O. Mayer et al, Arzneim. Forsch.,21(2) , 298-303 (1971)]. These cycloserine isomers have also beenevaluated for psychological and physiological effects in human subjects.For example, D-cycloserine when administered at 500 mg/day doses tohealthy human subjects, appeared to stimulate slight sociability, butwith depressed mental alertness [M. Vojtechovsky, Act. Nerv. Super.,7(3) 269 (1965)]. Also, D-cyloserine has been administered at 1000 to1500 mg/day to healthy volunteers whose blood levels showed increasedlevels of monoamine oxidase enzyme activity [V. Vitek et al,Psychopharmacologia, 7(3), 203-219 (1965)].

D-cycloserine has been investigated as a therapeutic agent for mentaldisorders in clinical trials, wherein D-cycloserine was administered tomentally disturbed patients at doses of 500 mg. per day [G. E. Crane,Compr. Psychiat., 2, 51-53 (1961)]. In such clinical trials,improvements in depression, insomnia, anexoria or tension were found forsome patients, while patients suffering from severe neurosis orpsychosis responded poorly to such medication. Moreover, D-cycloserinehas been used to exacerbate the symptoms of schizophrenia in an attemptto cure the ailment by symptom provocation [J. Simeon et al, Compr.Psychiat., 11, 80-88, (1970)]. It appears that D-cycloserine, at thedose levels used in these studies, is acting as an antagonist at theglycine site of the NMDA-PCP receptor complex mimicking the action ofPCP by inducing psychosis.

D-cycloserine has been sold commercially for treatment againstMycobacterium tuberculosis. When used at tuberculostatic doses,D-cycloserine is accompanied by many adverse side effects. The mostfrequent adverse side effects known involve the nervous system. In fact,the limiting factor in use of cycloserine is its CNS toxicity, includingboth neurologic and psychic disturbances [Drug Evaluation, Chapter 75,American Medical Association, Chicago (1986)]. Patients receivingD-cycloserine have been noted to suffer from drowsiness, dizziness,headache, lethargy, depression, tremor, dysarthria, hyperreflexia,paresthesia, nervousness, anxiety, vertigo, confusion and disorientationwith loss of memory, paresis, major and minor clonic seizures,convulsions and coma [G. K. McEvoy et al, American Hospital FormularyService: Drug Information, 8:16, American Society of HospitalPharmacists, Bethesda, Md. (1986)].

Other side effects have also been associated with treatments usingD-cycloserine. In chronic administration of tuberculostatic doses topatients in clinical trials, D-cycloserine has been observed to produceepisodes of diarrhea and oral mucositis. Diarrhea episodes are believedto be linked to depletion of natural intestinal flora by D-cycloserineinterference with flora cellular production. Several attempts have beenmade to reverse this flora depletion effect associated withD-cycloserine treatments. For example, the antibacterial effect ofD-cycloserine on Mycobacterium paratuberculosis has been reversed bymycobactin [W. B. Sutton et al, Antibiot. Chemotherapy, 5, 582-584(1955)]. Patients under treatment with tuberculostatic doses ofD-cycloserine, and suffering from diarrhea, have been given preparationsof Streptococcus faecium which reduced significantly the episodes ofdiarrhea [M. Borgia et al, Curr. Therap. Res., 31, 2, 265-271 (1982)].It is also a well-known remedy to use certain aged, fermented cheeses,such as Camembert or Maroilles cheese, to restore flora depleted byantibiotic treatment.

The growth-inhibiting effect of D-cycloserine on bacteria has been shownto be competitively reversed by D-alanine, a compound noted to be astructural analogue of D-cycloserine [J. W. Moulder et al, J.Bacteriol., 85, 707-711 (1962)]. It has been found that D-cycloserine,as a competitive inhibitor of alanine racemase, is bound to the alanineracemase enzyme 100 times more effectively than the natural substrateD-alanine [U. Roze et al, Mol. Pharmacol., 2, 92-94 (1966)].

Other interactions between D-cycloserine and alanine-type compounds areknown. For example, U.S. Pat. No. 4,031,231 describes antibacterialcompositions containing 3-fluoro-D-alanine-type compounds, such as3-fluoro-D-alanine and its deutero analogues, in combination with a3-fluoro-D-alanine autoantagonist-inhibitor, such as D-cycloserine.These compositions are described as having synergistic antibacterialaction.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a graph showing concentration of D-cycloserine influence onmaximal glycine stimulation of TCP binding in the presence of variousconcentrations of glycine.

FIG. 2 is a graph showing concentration of glycine influence on maximalglycine stimulation of TCP binding in the presence of variousconcentrations of D-cycloserine.

DESCRIPTION OF THE INVENTION

A therapeutic method for improvement of cognitive function or treatmentof a cognitive dysfunction or a psychotic disorder is achieved bytreatment of a subject, when such therapy is indicated, with acombination therapy of a therapeutically-effective amount of acycloserine-type compound and a therapeutically-effective amount ofD-alanine. The phrase "combination therapy", as used herein, is intendedto embrace administration of the cycloserine and D-alanine components ina sequential manner or to embrace co-administration of these twocomponents in a simultaneous manner. Co-administration of these twocomponents for cognitive-function improvement or cognitive-dysfunctiontreatment or antipsychotic treatment, may be accomplished with apharmaceutical composition having as active components atherapeutically-effective amount of a cycloserine-type compound and atherapeutically-effective amount of D-alanine. Preferably thiscomposition will contain one or more pharmaceutically-acceptableexcipients. More preferred is a pharmaceutical composition consistingessentially of a therapeutically-effective amount of a cycloserinecompound and a therapeutically-effective amount of D-alanine. Atherapeutically-effective amount of D-alanine is defined as aside-effect suppressing amount of D-alanine. Examples of adverseside-effects which can be prevented or reduced by D-alanineadministration are gastro-intestinal related distresses such as diarrheaand destruction of the intestinal flora.

A preferred type of cycloserine compound is D-cycloserine. Thepharmaceutical composition should contain D-cycloserine and D-alanine ina therapeutically-effective ratio.

The phrase "therapeutically-effective ratio" embraces a range ofrelative amounts of D-alanine and D-cycloserine which will be effectiveto improve cognitive dysfunction or to treat psychosis, while at thesame time being effective to reduce adverse side effects associated withuse of D-cycloserine alone. Improvement in cognitive function meansgenerally improvement in memory or learning ability. Treatment ofcognitive dysfunction includes treatment of neurodegenerative diseasessuch as Alzheimer's disease, age-associated memory impairment or alearning deficit. It is believed that a psychotic disorder is linked toan increased concentration of an endogenous ligand acting at the PCPsite of the NMDA-PCP receptor complex. This endogenous ligand isbelieved to be PCP-like in character in that interaction of the ligandwith the NMDA-PCP receptor complex results in inhibition of the openingof the ion channel triggered by NMDA. A Glycine B agonist compound ofthe invention, by potentiating NMDA transmission, will thus antagonizethe effect of the endogenous ligand. Inasmuch as the endogenous ligandis responsible for psychotic disorders, such as schizophrenia, theblocking of such ligand action should result in reduction of psychoticbehavior. In particular, it is believed that the compounds of theinvention will be useful in the treatment of acute or chronicPCP-induced psychosis.

A therapeutically-effective ratio of D-alanine to D-cycloserine would bein a range from about 1-to-1 to about 100-to-1.

D-cycloserine is 4-amino-3-isoxazolidone having the structural formula##STR1## This compound exists in the L- and D-isomeric forms, of whichthe compound D-cycloserine is more highly preferred.

Also embraced by this invention are the tautomeric forms of theforegoing cycloserine compounds as represented by ##STR2##

Included within the family of compounds of this invention are theisomeric forms of the described compounds including diastereoisomers,and the pharmaceutically-acceptable salts thereof. The term"pharmaceutically-acceptable salts" embraces salts commonly used to formalkali metal salts and to form addition salts of free acids or freebases. Since the cycloserine compounds contain basic nitrogen atoms,such salts are typically acid addition salts or quaternary salts. Thenature of the salt is not critical, provided that it is pharmaceuticallyacceptable, and acids which may be employed to form such salts are, ofcourse, well known to those skilled in this art. Examples of acids whichmay be employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, sulphuric acid andphosphoric acid, and such organic acids as maleic acid, succinic acidand citric acid. Other pharmaceutically acceptable salts include saltswith alkali metals or alkaline earth metals, such as sodium, potassium,calcium and magnesium, or with organic bases, such as dicyclohexylamine.All of these salts may be prepared by conventional means by reacting,for example, the appropriate acid or base with the correspondingcycloserine compound.

Cycloserine compounds for use in the invention may be synthesized bymethods described in the literature. For example, syntheses of N-acylderivatives and Schiff-base derivatives of D-cycloserine are describedby N. P. Jensen et al, J. Med. Chem., 23 6-8 (1980). Syntheses ofN,N'-diacyl derivatives of cycloserine are described by J. C. Howard, J.Org. Chem., 46, 1720-1723 (1981). . Syntheses of alkyl derivatives ofcycloserine are described by C. H. Stammer, J. Med. Chem., 13(6), 1013(1970). Syntheses L- and D-isomers of cycloserine, as well as analoguesthereof, are described by Pl. A. Plattner et al, Helv. Chim. Acta., 401531 (1957). There are many commercial sources of D-alanine, as well asmany published methods for making D-alanine.

BIOLOGICAL EVALUATION Glycine Binding Assay Procedure

Synaptic plasma membranes (SPM) were prepared from rat forebrain andstored as previously described [J. B. Monahan and J. Michel, J.Neurochem., 48, 1699-1708 (1987)]. Frozen membranes were thawed anddiluted 1:20 with 0.04% triton X-100 in 50 mM tris/acetate (pH 7.4).Following incubation at 37° C. for 30 min., the SPM were collected bycentrifugation at 95,000 X g for 15 min. The pellet was resuspended in50 mM tris/acetate (pH 7.4, triton-free) and hand-homogenized fivetimes. The membranes were again centrifuged as above. The pellet waswashed two additional times with 50 mM tris/acetate (withouthomogenization) and centrifuged. The final pellet was resuspended withhomogenization in 50 mM tris/acetate.

In the general receptor binding assay procedure, 10 nM [³ H]glycine wasadded to the appropriate concentration of the test compounds and theassay initiated by the addition of 0.2-0.4 mg of ice cold SPM. Theassay, which was done in 1.5 ml centrifuge tubes, was adjusted to atotal volume of 1.0 ml with all additions being made in 50 mMtris/acetate, pH 7.4 at 4° C. After a 10 minute incubation at 2° C., thesamples were centrifuged for 15 min. at 12,000 g (4° C.) in a BeckmanMicrofuge 12. The supernatant was aspirated and the tube tip containingthe pelleted membranes cut off and agitated in 0.5 ml of Beckman BTS-450tissue solubilizer for a minimum of 6 hours at room temperature. BeckmanMP scintillation cocktail (5 ml) containing 7 ml/liter acetic acid wasthen added and the samples counted on a Beckman LS 5800 liquidscintillation counter with automatic corrections for quenching andcounting efficiency. Nonspecific binding was defined as the residualbinding in the presence of 0.1 mM glycine and usually amounted to 25-35%of the total binding The binding of [³ H]glycine to the SPM was analyzedusing Scatchard and Hill transformations and the K_(i) for othercompounds was determined using logit-log analysis. Calculations andregression analysis were performed using templates developed for Lotus123 as previously described.

    ______________________________________                                        Result         K.sub.i (μM)                                                ______________________________________                                        Glycine        0.18                                                           D-cycloserine  1.92                                                           L-cycloserine  >100                                                           ______________________________________                                    

TCP Modulation Assay

[³ H]TCP binding was performed using Triton X-100 washed synaptic plasmamembranes (SPM) prepared from rat forebrain (30-45 day old, maleSprague-Dawley; Sasco, St. Charles, Mo.) as described previously [J. W.Thomas, W. F. Hood, J. B. Monahan, P. C. Contreras and T. L. O'Donohue,Brain Res., 442, 396-398 (1988)]. The assay was initiated by theaddition of SPM (0.15-0.25 mg) to an incubation containing 2.0 nM [³H]TCP (47.1 Ci/mmole; New England Nuclear, Boston, Mass.) and variousconcentrations of the appropriate test compound in a total volume of 0.5ml (all additions were made in 5 mM Tris/HC1 buffer, pH 7.4) andcontinued for 60 min at 25° C. The samples were then filtered thoughglass fiber filters (Schleicher and Schuell #32) which were pretreatedwith 0.05% (v/v) polyethylenimine. The filters were washed and theradioactivity quantitated by liquid scintillation spectrometry.Stimulation of [³ H]TCP binding was measured as an increase in basalspecific binding (basal binding=2583 ±381 DPM and this value increasedto a maximum of 4712±779 DPM in the presence of 0.6 μM glycine) withnonspecific binding as the residual binding in the presence of 60 μM PCP(562±30 DPM). The K_(d) for [³ H]TCP under basal conditions was 44 nM.The EC₅₀ values for the stimulation of [³ H]TCP binding were determinedusing a four parameter logistic regression analysis.

D-Cycloserine stimulates basal [³ H]TCP binding in a dose dependentmanner with an EC₅₀ =19.7 μM. Previous data show that D-cycloserineinteracts with the NMDA-associated [³ H]glycine recognition site (K_(i)=2.33±0.29 μM). No affinity for the NMDA recognition site, however, wasdetected as evidenced by the lack of displacement of NMDA-specific L-[³H]glutamate binding (K_(i) >100 μM). This finding indicates thatD-cycloserine enhances [³ H]TCP binding through its interaction with theNMDA receptor-associated glycine recognition site (herein defined as the"Glycine B receptor"). The maximal stimulation produced byD-cycloserine, however, was significantly less than that produced byboth glycine and D-serine.

This apparent lower efficacy indicates the potential partial agonistcharacter of D-cycloserine which was confirmed by the followingexperiment. As shown in FIG. 1, in the absence of exogenously addedglycine, D-cycloserine has agonist properties and stimulates [H]TCPbinding to a maximum of 40-50% of the stimulation induced by glycinealone. However, in the presence of various concentrations of glycine(0.1-0.6 μM), D-cycloserine has an apparent antagonist character andreduces the maximal level of glycine stimulation. These data provide afamily of D-cycloserine dose-response curves (generated in the presenceof several fixed concentrations of glycine) which asymptoticallyapproach 40-50% of the maximal stimulation induced by glycine alone, apattern characteristic of compounds with partial agonist properties asis known with different compounds acting on other receptors.

Further confirmation of the partial agonist character of D-cycloserinewas demonstrated in experiments wherein a glycine dose-response analysiswas performed in the presence of several fixed concentrations ofD-cycloserine (0-100 μM). As shown in FIG. 2, D-cycloserine potentiatedthe glycine stimulation of [³ H]TCP binding at glycine concentrationsbelow 0.1 μM, while at higher glycine concentrations (0.1-15 μM)D-cycloserine produced a rightward shift in the dose-response curve.These results are again consistent with partial agonist characteristics.

The functional analysis of D-cycloserine described herein is the firstreport of a compound interacting at this glycine modulatory siteexhibiting partial agonist characteristics. These results along with thefavorable brain bioavailability of the compound and evidence forinvolvement of the NMDA receptor in learning and memory potentially makeD-cycloserine a valuable tool to probe NMDA receptor function. Moreimportantly, Glycine B partial agonists would be expected to providetherapeutic benefits in treatment of psychosis, cognitive dysfunctions,such as Alzheimer's Disease, age-associated memory impairment,multi-infarct dementia, mixed organic brain syndrome metabolicencephalopathies of various origins, alcoholic dementia and variouslearning disorders. In particular, the Glycine B partial agonistcompounds would be useful in treatment of schizophrenia, Alzheimer'sDisease, age-associated memory impairment and learning deficit, in humansubjects suffering from such disorders, as well as for use inimprovement of memory and learning ability in healthy individuals.

PASSIVE AVOIDANCE ASSAY METHODS

Subjects: Male Long-Evans rats weighing about 200 g (Sasco) were used.They were housed two per cage with ad lib food and water for theduration of the experiment.

Apparatus: The apparatus consisted of a plexiglass box (32×26×20 cm)with a lid with a floor of metal rods spaced 1.8 cm apart. The box wasdivided into two chambers, one painted black and the other gray. Twodoors (12 cm high) were cut into the front of the box allowing accessinto each chamber.

A Y-shaped plexiglas runway was attached to the front of the box. Thestem of the Y was 16 cm long and unpainted. The arms of the Y (14 cmlong each) led to the two doors and each was painted the color of thechamber to which it led. The stem of the Y extended over the edge of thetable on which the apparatus was placed, so that it was approximately 75cm above the floor. The metal floor of the box was wired to a Lafayetteshock generator so that a 0.5 mAmp shock could be delivered.

Procedure: On the first rest day, each rat was placed on the runway andallowed to enter one of the chambers. The door to this chamber was thenclosed, and the rat was then allowed to enter the other chamber. On thesecond test day, some of the rats were given i.p. injections of eitherD-cycloserine dissolved in 0.9% saline, or saline alone. Sixty minuteslater, each rat was again allowed to enter one chamber, where itreceived a footshock for 2 seconds. If the rat did not previouslyreceive an injection, it was injected with either D-cycloserine orsaline ten seconds after the footshock. On the third test day, the ratis again placed on the runway and allowed to enter a chamber. On daystwo and three, each rat's latency to enter a chamber, and which chamberit entered, are recorded.

Effects of D-cycloserine (10 mg/kg i.p.) on passive avoidance learninglatency (secs.) to enter box 24 hours after shock are shown in Table I.

                  TABLE I                                                         ______________________________________                                                    Time of Drug Treatment                                                        Before Shock                                                                           After Shock                                              ______________________________________                                        Saline        8.9 ± 1.5                                                                             14.8 ± 3.1                                                      (n = 6)    (n = 5)                                              D-cycloserine 16.6 ± 3.0                                                                            22.8 ± 2.4                                                      (n = 6)    (n = 6)                                              ______________________________________                                    

In this animal model for demonstrating memory enhancement, the delay intime for the rat to enter the chamber (the "latency period") is ameasure of the rat's memory of the previous experience in receiving afoot shock. The longer is the latency period, the better is the memoryenhancing effect of the tested compound. Those animal experiments showthat D-cycloserine acting as a glycine ligand has memory-enhancingeffect which is characterized in this model by an increased latency forthe animal to enter the compartment.

The dose-effect relationship of D-cycloserine, as well as the effect ofthis compound when administered just before the information retrievaltrial, were also studied, as reported in Table II.

                  TABLE II                                                        ______________________________________                                        % of Saline Control Latency in Passive Avoidance                              Dose of DCS                      Administration                               (mg/kgi.p.)                                                                            pre-shock (%)                                                                             post-shock (%)                                                                            pre-retrieval                                ______________________________________                                        Saline   100         100         100                                          0.3      152*        160*        105                                          3        245*        215*         153*                                        10       138*        153*         180*                                        20                               100                                          ______________________________________                                         *Statistically different from control (P<0.05, ttest)                    

The effect of co-administration of D-cycloserine and D-alanine to ratswas investigated following the methodology described for Table II.Results are shown in Table III.

                  TABLE III                                                       ______________________________________                                        Latency in Seconds in Passive Avoidance                                                          Dose       Latency                                         Compound           (mg/kg I.G.)                                                                             (sec.)                                          ______________________________________                                        Saline                        11.9                                            D-alanine          534        10.9                                            D-cycloserine       60        17.3*                                           D-alanine + D-cycloserine                                                                        534 + 60   17.4*                                           ______________________________________                                         *Statistically different from control (P<0.05, ttest)                    

These results in Table III show that the memory enhancement effect ofD-cycloserine is not altered when D-cycloserine is co-administered withD-alanine.

Rewarded Alternation of Rats in a T-maze

Rats were trained on a place learning task in a T-maze following i.p.administration of D-cycloserine (3 mg/kg) or saline. Both groups learnedthe task in about 20 trials. Learning is defined as making 9 correct outof 10 consecutive choices. On the following day, the food reward wasplaced in the other arm of the maze (reversal). The saline-treated ratsrequired about 32 trials to learn the reversal, while the D-cycloserinerats learned the reversal in about 20 trials. These data confirm in thisbehavioral paradigm that D-cycloserine has a facilitating effect onprocesses of learning and memory.

Intact hippocampal structure is necessary for the brain to processinformation and store it in memory. The phenomenon of "long termpotentiation" (LTP) seems to be the mechanism by which this processoccurs. The leading role of the N-methyl-D-aspartate ("NMDA") receptor,a sub-type of excitatory amino acid receptor, in LTP has been firmlyestablished by electrophysiological studies. NMDA antagonists such as2-amino-7-phosphonoheptanoic acid (APH) inhibit the establishment orpropagation of LTP.

Recently, it has been demonstrated in neurophysiological studies thatglycine potentiates the response to activation of NMDA receptors incultured brain neurons. This is a strychnine-insensitive action and itis postulated to result from activation of a supraspinal glycinereceptor (herein defined as the Glycine B receptor) which modulates theopening of the Na⁺ -Ca⁺⁺ channel triggered by NMDA activation. Forexample, milacemide, as a glycine prodrug, increases the whole braincontent of glycine by 30%. By the mechanism explained above, thisincrease of glycine can lead to a facilitation of NMDA transmission andimproved memory and learning ability.

Data presented in Table II demonstrate that the dose-effect relationshipof D-cycloserine is characterized by a bell-shaped curve. The unusualrelationship is believed to be associated with the partial agonistcharacter of D-cycloserine. It is apparent, therefore, that maximumefficacy will be achieved within a defined range and that higher dosese.g., greater than 500 mg per dose in human subjects, would be expectedto result in reduced efficacy.

The acidic amino acids, aspartic and glutamic acid, have been found topossess both excitatory and excitotoxic properties [J. W. Olney,Science, 164, 719-721 (1969); J. W. Olney et al., Exp. Brain Res., 14,61-76 (1971)]. Indeed, neurons which have excitatory amino acidreceptors on their dendritic or somal surfaces undergo acute excitotoxicdegeneration when these receptors are excessively activated by glutamicacid.

Glycine agonists which have a potentiating effect on the NMDAtransmission would be expected to increase the glutamic acidexcitotoxicity. A Glycine B partial agonist achieves beneficialexcitatory effects without the detrimental excitotoxic side effect Mostglycine ligands are very polar molecules and hardly cross the bloodbrain barrier. Because of the difficulty in crossing the blood brainbarrier, such ligands are not bioavailable at concentrations effectiveto be therapeutically beneficial. It is known that D-cycloserine easilypasses the blood brain barrier [Goodman and Gilman, The PharmacologicBasis of Therapeutics, Ch., 53, 1210-1211 (1980)]. It was surprising andunexpected that D-cycloserine was found to have such a good affinity forthe strychnine-insensitive glycine receptor as shown by the binding dataabove. Glycine agonists are believed to facilitate NMDA transmissionand, therefore, to have a positive effect on LTP. The improvement in LTPis postulated to be linked to memory enhancement. Such glycine agonistsare also believed to have potential for reversing the symptoms ofschizophrenia and, in particular, to reverse the symptoms induced byacute or chronic PCP intoxication.

Sequential or co-administration of a cycloserine compound and D-alaninemay be achieved by any technique capable of introducing the combinationof compounds into the gastrointestinal system.

Such combinations indicated for prophylactic therapy will preferably beadministered in a ratio range from about 1:1 to about 100:1 of D-alanineto the cycloserine compound. Preferably, the ratio of D-alanine toD-cycloserine will be in a range from about 10:1 to about 100:1. Ingeneral, such combinations may be administered based upon the dose ofD-cycloserine effective to enhance memory or treat cognitivedysfunction. Such effective amount of D-cycloserine will generally be ina daily dose in a range from about 0.01 mg to about 10 mg per kilogramof body weight per day. A more preferred dosage will be a range fromabout 0.01 mg to about 5 mg per kilogram of body weight. Most preferredis a dosage in a range from about 0.05 to about 2.5 mg per kilogram ofbody weight per day. A suitable dose can be administered in multiplesub-doses per day. These sub-doses may be administered in unit dosageforms. Typically, a dose or sub-dose may contain from about 1 mg toabout 100 mg of active compound per unit dosage form. A more preferreddosage will contain from about 2 mg to about 50 mg of active compoundper unit dosage form. Most preferred is a dosage form containing fromabout 3 mg to about 25 mg of active compound per unit dose.

The active compounds are usually administered in apharmaceutically-acceptable formulation. Such formulations may compriseeffective amounts of each active compound together with one or morepharmaceutically-acceptable carriers or diluents. Other therapeuticagents may also be present in the formulation. Apharmaceutically-acceptable carrier or diluent provides an appropriatevehicle for delivery of the active compounds without introducingundesirable side effects. Delivery of the active compounds in suchformulations may be by various routes including oral, nasal, topical,buccal and sublingual.

Formulations for oral administration may be in the form of capsulescontaining the active compounds dispersed in a binder such as gelatin orhydroxypropylmethyl cellulose, together with one or more of a lubricant,preservative, surface-active or dispersing agent. Such capsules ortablets may contain a controlled-release formulation as may be providedin a disposition of active compounds in hydroxypropylmethyl cellulose.

Although this invention has been described with respect to specificembodiments, the details of these embodiments are not to be construed aslimitations. Various equivalents, changes and modifications may be madewithout departing from the spirit and scope of this invention, and it isunderstood that such equivalent embodiments are part of this invention.

What is claimed is:
 1. A therapeutic method for treating Alzheimer'sDisease in an individual when such therapy is indicated, comprisingadministering to the individual a therapeutically effective amount ofD-cycloserine and a therapeutically-effective amount of D-alanine, saidD-cycloserine and said D-alanine being in a therapeutically-effectiveratio, said therapeutically-effective ratio being in a range of about1-to-1 to about 100-to-1 of said D-alanine to said D-cycloserine, saidD-cycloserine being administered in an amount in a range from 1 mg toabout 100 mg of D-cycloserine per unit dosage form.
 2. The method ofclaim 1 wherein said D-cycloserine is administered in an amount in arange from about 2 mg to about 50 mg of D-cycloserine per unit dosageform.
 3. The method of claim 2 wherein said D-cycloserine isadministered in an amount in a range from about 2 mg to about 50 mg ofD-cycloserine per unit dosage form.
 4. A therapeutic method for treatingage-associated memory impairment in an individual when such therapy isindicated, comprising administering to the individual a therapeuticallyeffective amount of D-cycloserine and a therapeutically-effective amountof D-alanine, said D-cycloserine and said D-alanine being in atherapeutically-effective ratio, said therapeutically-effective ratiobeing in a range of about 1-to1 to about 100to1 of said D-alanine tosaid D-cycloserine, said D-cycloserine being administered in an amountin a range from 1 mg to about 100 mg of D-cycloserine per unit dosageform.
 5. The method of claim 4 wherein said D-cycloserine isadministered in an amount in a range from about 2 mg to about 50 mg ofD-cycloserine per unit dosage form.
 6. The method of claim 5 whereinsaid D-cycloserine is administered in an amount in a range from about 2mg to about 50 mg of D-cycloserine per unit dosage form.
 7. Atherapeutic method for treating learning deficit in an individual whensuch therapy is indicated, comprising administering to the individual atherapeutically effective amount of D-cycloserine and atherapeutically-effective amount of D-alanine, said D-cycloserine andsaid D-alanine being in a therapeutically-effective ratio, saidtherapeutically-effective ratio being in a range of about 1-to1 to about100-to-1 of said D-alanine to said D-cycloserine, said D-cycloserinebeing administered in an amount in a range from 1 mg to about 100 mg ofD-cycloserine per unit dosage form.
 8. The method of claim 7 whereinsaid D-cycloserine is administered in an amount in a range from about 2mg to about 50 mg of D-cycloserine per unit dosage form.
 9. The methodof claim 8 wherein said D-cycloserine is administered in an amount in arange from about 2 mg to about 50 mg of D-cycloserine per unit dosageform.
 10. A therapeutic method for improving memory or learning abilityin a healthy individual, comprising administering to the individual atherapeutically effective amount of D-cycloserine and atherapeutically-effective amount of D-alanine, said D-cycloserine andsaid D-alanine being in a therapeutically-effective ratio, saidtherapeutically-effective ratio being in a range of about 1-to-1 toabout 100-to-1 of said D-alanine to said D-cycloserine, saidD-cycloserine being administered in an amount in a range from 1 mg toabout 100 mg of D-cycloserine per unit dosage form.
 11. The method ofclaim 10 wherein said D-cycloserine is administered in an amount in arange from about 2 mg to about 50 mg of D-cycloserine per unit dosageform.
 12. The method of claim 11 wherein said D-cycloserine isadministered in an amount in a range from about 2 mg to about 50 mg ofD-cycloserine per unit dosage form.
 13. A therapeutic method fortreating a psychotic disorder in an individual when such therapy isindicated, comprising administering to the individual a therapeuticallyeffective amount of D-cycloserine and a therapeutically-effective amountof D-alanine, said D-cycloserine and said D-alanine being in atherapeutically-effective ratio, said therapeutically-effective ratiobeing in a range of about 1-to-1 to about 100-to-1 of said D-alanine tosaid D-cycloserine, said D-cycloserine being administered in an amountin a range from 1 mg to about 100 mg of D-cycloserine per unit dosageform.
 14. The method of claim 13 wherein said D-cycloserine isadministered in an amount in range from about 2 mg to about 50 mg ofD-cycloserine per unit dosage form.
 15. The method of claim 14 whereinsaid D-cycloserine is administered in an amount in a range from about 2mg to about 50 mg of D-cycloserine per unit dosage form.
 16. The methodof claim 13 wherein said psychotic disorder is a schizophrenic disorder.17. The method of claim 16 wherein said schizophrenic disorder is aPCP-induced schizophrenic disorder.